Asymmetric cell division, a process by which a cell divides to produce two different daughter cells, is essential for generating cellular diversity during development. Such divisions are also an attractive means for stem cells to balance the competing needs of self-renewal and differentiation during organogenesis and tissue maintenance, by producing one daughter that remains as a stem cell and the other that differentiates. In Drosophila, Numb, a cytoplasmic signaling protein, localizes asymmetrically in dividing precursor cells and segregates primarily into one daughter cell. This asymmetric Numb inheritance is essential for the two daughter cells to adopt different fates. We have been using the mammalian numb proteins, Numb and Numbl, as an entry point, and neurogenesis in mice as a model system, to probe the contribution of two modes of cell division - symmetric vs. asymmetric - in regulating the behavior of stem cells. Neurons in the mammalian central nervous system are generated over an extended period of time during development, requiring neural stem (progenitor) cells to self-renew while generating neurons at the same time. We have postulated that neural progenitor cells balance self-renewal and differentiation during mouse neurogenesis by segregating Numb and Numbl asymmetrically to promote progenitor over neuronal fates in asymmetric divisions that produce another progenitor and a neuron. Indeed, neural progenitor cells lose their ability to self-renew in the absence of Numb and Numbl, whereas forcing them to segregate numb symmetrically inhibits neuron production by forcing their daughter cells to both choose self-renewal over differentiation. We have also identified an essential Numb and Numbl partner, ACBD3, and a novel mechanism that regulates the timing of numb activity in cell-fate specification by using the process of Golgi fragmentation and reconstitution during cell cycle to change the subcellular distribution of ACBD3. Here we seek to address how numb asymmetric localization and numb activity are regulated by combining studies using mice and Drosophila. We hypothesize that the essential regulators of numb signaling are evolutionarily conserved but differentially used in mice and Drosophila to meet their specific demands of neurogenesis. We intend to elucidate how mammalian numb proteins are asymmetrically segregated by neural progenitor cells, by first examining different Numbl protein variants for their ability to mediate asymmetric cell division during mouse neurogenesis and then identifying and characterizing the proteins binding specifically to the Numbl variant that segregates asymmetrically. Mammalian numb and ACBD3 proteins, when introduced into Drosophila, can act synergistically in a dosage-dependent manner to specify cell fates. Thus, we seek to identify novel components of the numb signaling pathway by performing a genetic screen in Drosophila to search for enhancers and suppressors of Numb-ACBD3 activity. We also seek to determine the function of the Drosophila ACBD3 homologue. Through the proposed studies, we hope to achieve a better understanding of how numb proteins specify neural progenitor fates and use the knowledge as entry points to elucidate the essential mechanisms that regulate the behavior of neural stem cells in mammals. A better understanding of how stem cells are regulated will provide novel insights for their therapeutic use to repair tissues damaged by disease, injury or aging. PUBLIC HEALTH RELEVANCE: We seek to identify novel regulators of numb proteins, which are evolutionarily conserved signaling molecules that segregate asymmetrically to allow the two daughter cells to adopt different fates after an asymmetric cell division. We have been using the mammalian numb proteins, Numb and Numbl, to probe the contribution of two modes of cell division - symmetric vs. asymmetric - by stem cells during neurogenesis in mice. Our studies have revealed an essential requirement for numb-mediated asymmetric cell division in allowing neural stem/progenitor cells to balance self-renewal and differentiation as well as a previously unrecognized homeostasis mechanism that strictly controls the number of stem cells during neurogenesis. Whereas asymmetric division allows stem cells to balance self-renewal and differentiation during organogenesis and tissue maintenance, stem cells can use symmetric divisions to expand their population, for example, in response to tissue injury. It is also likely that cancer stem cells are sustained by self-renewing symmetric divisions. Thus, a better understanding of numb function may yield novel insights for achieving a key goal of stem-cell research, which is to repair tissues damaged by disease, injury or aging by introducing stem cells from external sources or expanding the endogenous populations.